Red blood cell substitute emulsions containing alkyl- or alkylglyucerophosphoryl choline surfactants and methods of use

ABSTRACT

Oil and water emulsions containing alkylphosphoryl choline or alkylglycerophosphoryl choline surfactants are disclosed. The surfactants have the following general structures: ##STR1## In the above general structures, R 1 , R 2  or R 3  is alkyl, alkenyl, fluoroalkyl and alkenyl; and PC is the phosphoryl choline. The emulsions are useful as oxygen transport agents, artificial bloods or red blood cell substitutes.

This is a division of application Ser. No. 07/791,420, filed Nov. 13,1991, now U.S. Pat. No. 5,304,325.

TECHNICAL FIELD OF THE INVENTION

This invention relates to emulsions of oil and water and processes ofmaking and using them. More particularly, this invention relates tonovel emulsions that contain an oil (using this term in a generalsense), water and a novel surfactant that may be generally identified asan alkylphosphoryl choline or an alkylglycerophosphoryl choline. Suchemulsions have general utility for many industrial uses and areespecially useful as oxygen transport agents, artificial bloods or redblood cell substitutes and as contrast agents for biological imaging.

BACKGROUND OF THE INVENTION

Emulsions permit extensive subdivision of an oil (using this term in ageneral sense) with a consequent formation of an enormous oil-waterinterface. Emulsions may be of the oil-in-water type or water-in-oiltype and may involve other phases and interfaces. Where thesedispersions are of the oil-in-water type, they are usually referred toas oil emulsions or soluble oil compositions. Where the phases arereversed as in a water-in-oil type, they are commonly referred to asinverted emulsions. There are many industrial uses of oil and wateremulsions including paints, adhesives, lubricants, cleansing agents,soaps, insecticides, cosmetics, pharmaceuticals and therapeutic agents.This short list is only the beginning of a vast number of uses for oiland water emulsions.

One class of emulsions that has developed over a number of years isfluorocarbon emulsions as oxygen transport agents or artificial bloods.U.S. Pat. No. 3,911,138 which issued to Clark is an early example fromthe patent art which discloses perfluorocarbon emulsions as artificialbloods. As developed in this patent, neat fluorocarbon liquids cannot beinjected into the blood stream, because their hydrophobic charactermakes them immiscible in the blood and, as a result, when they aretransported in small blood vessels, they may cause vascular obstructionand death. As a consequence, for medical uses that require intravascularinjection, highly fluorinated organic compounds or fluorochemicals mustbe dispersed as physiologically acceptable emulsions as developed in theabove Clark patent and U.S. Pats. Nos. 4,110,474; 4,187,252 and4,443,480.

There have been various attempts to make emulsions that are both stableand incorporate relatively large amounts of fluorocarbons that arerequired in clinical practice where the total volume of the emulsionthat can be administered is limited, e.g., as in artificial bloods. Anobjective in the preparation of such emulsions is the employment of anacceptable fluorocarbon that may be excreted from the body within aclinically acceptable time period. Furthermore, compositions arerequired that are sterilizable without destruction of their stability.

A fluorocarbon emulsion that has been approved by the FDA is FLUOSOL DAwhich is an emulsion of perfluorodecalin and perfluorotripropylamine ina mixture of two surfactants, namely, egg yolk phospholipid and PluronicF-68. This product, however, is not stable in the liquid state and mustbe stored frozen. Furthermore, the required presence of theperfluorotripropylamine in this emulsion to help stabilize itdisadvantages the emulsion's medical usefulness because the half life ofthe perfluorotripropylamine in the liver and other body tissues islonger than desirable (see K. Yokoyama et al, "A PerfluorochemicalEmulsion as an Oxygen Carrier" Artif. Organs (Cleve), 8 (1) pp. 34-40(1984)). Finally, this emulsion contains only about 12% fluorocarbon byvolume and thus it is much less therapeutically effective than desiredbecause of its low oxygen content capacity.

Various surfactants have also been investigated in an attempt to produceuseful and stable emulsions of fluorocarbons as oxygen transport agentsin artificial bloods. For example, fluorocarbon emulsions containing ahydrogenated phospholipid, a nonionic polymeric surfactant and asurfactant selected from C6-22 fatty acids, their salts andmonoglycerides, must also be stored at 4° C. See, e.g., Japanese Pat.Appln. 59-067,229; U.S. Pat. No. 4,252,827 and German Offen. DE 2630506.

European Pat. Appln. 87300454.3 of Clark and Shaw describes novelemulsions of highly fluorinated organic compounds for use as oxygentransport agents and artificial bloods. This Clark and Shaw applicationdiscloses emulsions that are stable even when they contain higher levelsof perfluorocarbons of up to about 75% by volume. The fluorocarbons ofthese emulsions display acceptable rapid excretion times from the liverand other body tissues, as well as being easily sterilized. Theseemulsions contain an oil as an emulsifying adjuvant in a compositioncontaining the fluorochemical, surfactant and water. While improvementsdisclosed in this Clark and Shaw application are significant andalleviate many of the difficulties in the long search for effectivetransport agents in artificial bloods, there is a continuing need forfurther development.

In brief, emulsions of the oil and water type provide a very importantrole in many industries and numerous patents have been granted coveringthem. Research continues with efforts toward developing new emulsifyingagents that provide emulsions having greater stability and broaderutility in many industries including medical and non-medical fields.

SUMMARY OF THE INVENTION

This invention is directed to emulsions of the oil and water type.According to this invention, novel surfactants have been found to formsurprisingly stable oil-in-water emulsions. More particularly,fluorochemical (hereinafter sometimes simply "PFC") emulsions have beenmade and found to significantly increase the circulatory blood residencetime of the PFC and favorably alter the tissue distribution of the PFCin critical organs, such as the liver and spleen. Furthermore, the novelsurfactants of this invention have been found to significantlyameliorate the adverse drop in hematocrit, or red blood cell count,after intravenous infusion, normally associated with most lecithin basedPFC emulsions.

The emulsions of this invention contain alkylphosphoryl choline oralkylglycerophosphoryl choline surfactants. These surfactants have thefollowing general structures: ##STR2##

More specifically, R₁, R₂ or R₃ is alkyl, alkenyl, fluoroalkyl andalkenyl. Each group may contain about 6 to about 54 carbon atoms, may besaturated or unsaturated, and may be a straight chain aliphatic group orbranched aliphatic group. C₁₂ -C₁₈ carbon atoms are presently preferredfor the R₁ and R₂ groups of general structure I, and C₆ -C₃₂ carbonatoms for R₃, when employed as surfactants for medical PFC emulsions ofthis invention. Most preferably, R₁ and R₂ are a C₁₄ -C₁₈ saturated orunsaturated aliphatic group. In general, these R groups are the residuesof aliphatic alcohol or halide reactants when synthesized by methods ofthis invention.

PC in the above structures is the phosphoryl choline group: ##STR3##where R₄ is hydrogen or lower alkyl such as methyl, ethyl and propyl.The hydrogen or methyl group is preferred in medical PFC emulsions ofthis invention.

This invention also includes methods of making these surfactants,emulsions containing them and methods of using them as oxygen transportagents, artificial bloods or red blood cell substitutes. Otherobjectives of this invention and advantages will become apparent fromthe following detailed description.

DETAILED DESCRIPTION

The emulsions of this invention comprise an oil, water and a surfactantof the above identified type. In general, the oil may be contained inamounts of from about 1 to 90% by total volume of the water and oil.More specifically, for instance, in medical applications for intravenoususe, about 60% v/v (115 w/v %) is a practical limit because of viscositylimitations for an intravenous product. Higher amounts may be employedfor other applications. The surfactant may be contained in amounts fromabout 0.5 to about 10% by weight, usually about 1-2% by weight of theemulsion. These components are identified with greater particularity asfollows.

A. Oil

The term "oil" is used herein in a general sense to identify a largeclass of substances whether of mineral, vegetable, animal, essential,synthetic or edible origin. Thus, the term "oil" is used herein asapplied to a wide range of substances that are quite different inchemical nature. In the classification of oils by type or function, forexample mineral oil is derived from petroleum and includes aliphatic orwax-based hydrocarbons, aromatic or asphalt-based hydrocarbons or mixedaliphatic and aromatic based hydrocarbons. Also included in the mineralclassification are petroleum-derived oils such as refined paraffin oil,and the like. In the vegetable classification, oils are chiefly derivedfrom seeds or nuts and include drying oils such as linseed and tongueoil; semidrying such as safflower and soy bean oils; nondrying such ascastor, cottonseed and coconut oils and inedible soap stocks such aspalm and coconut oils. In the animal classification, oils usually occuras fats in tallow, lard and stearic acid sources. The liquid animaltypes include fish oils, oleic acid, sperm oil, etc. and they usuallyhave a high fatty acid content. In the essential oil classification,complex volatile liquids may be derived from flowers, stems and leaves,and often the entire plant. These essential oils include turpenes suchas pinene, dipentene, etc. and are chiefly used for perfumery andflavorings. In the edible classification, included are some vegetableoils, such as olive, cottonseed, corn and peanut, as well as somespecial fish oils such as cod-liver, haliver, shark liver, and so forthwhich are used largely as medicines for their high vitamin content.Included in this wide range of substances that may be classified as oilsfor purposes of this description are those synthesized organic compoundsthat are oleophilic such as the highly fluorinated organic compounds orperfluorocompounds that are capable of being synthesized by well knownchemical or electrochemical processes.

In this description, "fluorochemical" or "PFC" is used to describeeither a highly fluorinated organic compound of perfluorocarbon orfluorinated chemical. Further, these terms are used interchangeably. Theterm "perfluorocarbon" includes a "cyclic" or "acyclic" compound ofcarbon. Substituted derivatives thereof are also included wherefluorocarbons have other elements within their structures such asoxygen, nitrogen and bromine, etc. It should also be noted that the term"perfluorocarbon" denotes substitution of all hydrogen atoms attached tothe carbon atom chain or ring and any carbon side groups with fluorine.However, "fluorocarbon" is meant to include partially or substantiallyfluorinated fluorinated compounds. This is permissible providing thatthe lack of complete replacement of all hydrogens does not affect theessential non-toxic characteristics of the preferred medicalfluorocarbons of this invention. Among the perfluorocarbon compoundswhich may be employed are perfluorotributylamine (FC47),perfluorodecalin (PP5), perfluoromethyldecal in (PP9),perfluorooctylbromide, per fluorotetrahydrofuran (FC80), perfluoroether(PID) [(CF₃)₂ CFOCF₂ (CF₂)₂ CF₂ OCF(CF₃)₂ ], perfluoroether(PIID)[(CF₃)₂ CFOCF₂ (CF₂)₆ CF₂ OCF(CF₃)₂ ], perfluoropolymer (E3)##STR4## perfluoropolymer (E4) ##STR5## perfluoroetherpolymer (FomblinY/01), perfluorododecane, perfluorobicyclo[4.3.0]nonane,perfluorotrimethylcyclohexane, perfluorotripropylamine,perfluoroisopropylcyclohexane, perfluoroendotetrahydrodicyclopentadiene,perfluoroadamantane, perfluoroexo-tetrahydrodicyclopentadiene,perfluorobicyclo[5.3.0.]decane, perfluorotetramethylcyclohexane,perfluoro-1-methyl-4-isopropylcyclohexane, perfluoro-n-butylcyclohexane,perfluorodimethylbicyclo[3.3.1.]nonane, perfluoro-1-methyl adamantane,perfluoro-1-methyl-4-t-butylcyclohexane, perfluorodecahydroacenaphthene,perfluorotrimethylbicyclo[3.3.1.]nonane, perfluoro-n-undecane,perfluorotetradecahydrophenanthrene,perfluoro-1,3,5,7-tetramethyladamantane, perfluorododecahydrofluorene,perfluoro-1,3-dimethyl adamantane, perfluoro-n-octylcyclohexane,perfluoro-7-methyl bicyclo[4.3.0.]nonane,perfluoro-p-diisopropylcyclohexane, andperfluoro-m-diisopropylcyclohexane. Chlorinated perfluorocarbons, suchas chloroadamantane and chloromethyladamantane as described in U.S. Pat.No. 4,686,024 may be used. Such compounds are described, for example, inU.S. Pats. Nos. 3,962,439; 3,493,581, 4,110,474, 4,186,253; 4,187,252;4,252,827; 4,423,077; 4,443,480; 4,534,978 and 4,542,147, European Pat.Applns. Nos. 80710 and 158,996, British Pat. Specification 1,549,038 andGerman Offen. 2,650,586. Of course, it should be understood thatmixtures of any of these highly fluorinated organic compounds may alsobe used in the emulsions and processes of this invention.

B. Surfactant

According to this invention, novel surfactants have been found to formsurprisingly stable oil-in-water emulsions. More particularly, stablePFC emulsions have been made and found to significantly increase thecirculatory blood residence time of the PFC and favorably alter thetissue distribution of the PFC in critical organs, such as the liver andspleen. Furthermore, surfactants of this invention have been found tosignificantly ameliorate the adverse drop in hematocrit, or red bloodcell count, after intravenous infusion, normally associated with mostlecithin based PFC emulsions.

The emulsions of this invention contain alkylphosphoryl choline oralkylglycerophosphoryl choline surfactants. These surfactants have thefollowing general structures: ##STR6##

More specifically, R₁, R₂ or R₃ is alkyl, alkenyl, fluoroalkyl andalkenyl. Each group may contain about 6 to about 54 carbon atoms, andmay be a straight chain aliphatic group or branched aliphatic group. C₁₂-C₁₈ carbon atoms are presently preferred for the R₁ and R₂ groups ofgeneral structure I, and C₆ -C₃₂ carbon atoms for R3, when employed assurfactants for medical PFC emulsions of this invention. Mostpreferably, R₁ and R₂ are a C₁₄ -C₁₈ saturated or unsaturated aliphaticgroup. In general, these R groups are the residues of aliphatic alcoholor halide reactants when synthesized by methods of this invention.

PC in the above structures is the phosphoryl choline group: ##STR7##where R₄ is hydrogen or lower alkyl such as methyl, ethyl and propyl.The hydrogen or methyl group is preferred in medical PFC emulsions ofthis invention.

Examples of aliphatic alcohol or halide reactants to form the R₁, R₂ orR₃ groups in the above structures are tetradecylbromide, oleyl alcohol,hexadecylbromide, octadecyl alcohol, 1H,1H,7H-dodecafluoro-1-heptanol,2-Iodo-1,1,1-trifluoroethane, 1H,1H,11H-eicosafluoroundecan-1-ol.,1-Iodo-1H-1H-perfluorobutane, 1-Iodo-1H,1H,2H,2H-perfluorodecane,1-Iodo-1H,1H,2H,2H-perfluorododecane,1-Iodo-1H,1H,2H,2H-perfluorooctane,4-Iodo-2-trifluoromethyl-1-1,1,1,2-tetrafluorobutane,2,2,3,3,4,4,5,5-octafluorohexan-1,6-diol,1-Iodo-1H,1H-pentadecafluoro-octanol-1, 3,3,4,4,4,-pentfluorobutanol-2,(1H,1H-pentafluoropropanol-1), and (Perfluoro-tert-butanol). Inaddition, Atochem, Dupont, 3M and Hoechst all market suitable mixturesof the formula R_(f) --CH₂ --CH₂ --X (X═OH,I) in which R_(f) iscomprised of varying mixtures of CnF_(2n+1) perfluoroalkyl linear andbranched chains. Typically, the mixture's "n" value is either 1-3, 4-8,or 6-12. These same companies sell R_(f) --COOH, and it is a trivialmatter to reduce the carboxylate in these compounds to produce R_(f) CH₂OH. Also marketed are perfluoroalkenes of many types (which could easilyserve as substrates for the addition of ROH) and oxiranes (the oxiranering is hydrogen containing, with the rest of the molecule beingperfluorochemical in nature). The oxirane, too, is easily openednucleophilically by ROH.

Specific examples of surfactants under the general structures are1,2-dioctylglycero-3-phosphoryl choline,1,2-ditetradecylglycero-3-phosphoryl choline,1,2-dihexadecylglycero-3-phosphoryl choline,1,2-di-octadecylglycero-3-phosphoryl choline,1-hexadecyl-2-tetradecylglycero-3-phosphoryl choline,1-octadecyl-2-tetradecylglycero-3-phosphoryl choline,1-tetradecyl-2-octadecylglycero-3-phosphoryl choline,1-hexadecyl-2-octadecylglycero-3-phosphoryl choline,1-2-dioctadecylglycero-3-phosphoryl choline,1-octadecyl-2-hexadecylglycero-3-phosphoryl choline,1-tetradecyl-2-hexadecylglycero-3-phosphoryl choline,2,2-ditetradecyl-1-phosphoryl choline ethane and1-hexadecyl-tetradecylglycero-3-phosphoryl choline and mixtures of thesenovel surfactants with other known surfactants may be employed asdescribed hereinafter.

C. Emulsion Characteristics

The emulsions of this invention are made by dispersing oil in water inthe presence of the above identified surfactants. The surfactantenhances the dispersion and stabilization of the liquid phases. Whiledispersions may be generally referred to herein as emulsions, it shouldbe understood that they may be considered solutions, micellar solutions,microemulsions, vesicular suspensions, or mixtures of all of thesephysical states. Accordingly, the term "emulsion" as used herein coversall these states and the novel surfactant or solubilizing agent isemployed to enhance stable mixtures of these physical states of the oiland water phases. For example, where a fluorochemical oil is emulsifiedin water, another oil may serve as an emulsifying adjuvant as describedin the aforementioned Clark and Shaw European Pat. Appln. 87300454.3. Wewish to emphasize that, according to this invention, this emulsifyingadjuvant is optional. Successful emulsions have been prepared with nosuch adjuvant. When an adjuvant is employed, a liquid fatty oil such asa mono-, di-, or triglyceride or mixtures thereof are the preferredagents. Where such oil and and oleophilic PFC combinations areemulsified in water, as provided hereinafter, complex phases andinterfaces may form.

Preferably, for artificial blood, the emulsions of this inventioncontain a PFC or mixture of PFCs, and most preferably contain afluorocarbon selected from the group consisting of perfluorodecalin,perfluoromethyldecalin, perfluorodimethyladamantane,perfluorooctylbromide, perfluoro-4-methyloctahydroquinolidizine,perfluoro-N-methyl-decahydroquinoline, F-methyl-1-oxa-decalin,perfluorobicyclo(5.3.0) decane, perfluoro-octahydroquinolidizine,perfluoro-5,6-dihydro-5-decene and perfluoro-4,5-dihydro-4-octene. Foruse as a contrast agent for biological imaging perfluorooctylbromide isone of the preferred PFCs according to this invention.

While the PFCs or mixture of PFCs may comprise 10% up to about 75% byvolume, or more, of the emulsions, preferably they comprise at least 40%by volume. When the emulsions are to be used as "artificial bloods" orred blood cell substitutes, the PFC is present in as high a volumeconcentration as possible, e.g., 40% by volume is often preferredbecause that concentration matches the approximate oxygen contentcapacity of whole blood.

In a broader sense, as indicated above, the amount of oil in theemulsions may vary over a wide range of concentrations from about 0.5 toabout 90% by volume, or more. It depends on the concentration andproperties of the other components of the emulsion and its use. Forexample, when used as an artificial blood, PFC oil is present in anacceptable amount along with other oil emulsifying adjuvant. The actualoil concentration to produce an acceptable emulsion for any given set ofcomponents is determined by preparing and testing the stabilities ofemulsions at various oil concentrations. Within this teaching for PFCartificial bloods, for instance, between 0 and 30% by weight oiladjuvant and 10-70% by volume PFC oil are used.

The amount of a particular surfactant used in the emulsions of thisinvention depends upon the amounts and properties of other components ofthe emulsion as indicated above. Generally about 0.5-10% by weight ofsurfactant, preferably, about 1-2% by weight is used. The surfactant ofthis invention may be used with other surfactants as indicated above.Among other surfactants useful in the emulsions of this invention areany of the known anionic, cationic, non-ionic and zwitter-ionicsurfactants. These include, for example, anionic surfactants, such asalkyl or aryl sulfates, sulfonates, carboxylates or phosphates, cationicsurfactants such as mono-, di-, tri- and tetraalkyl or aryl ammoniumsalts, non-ionic surfactants, such as alkyl or aryl compounds, whosehydrophilic part consists of polyoxyethylene chains, sugar molecules,polyalcohol derivatives or other hydrophilic groups and zwitter-ionicsurfactants that may be combination so the above anionic or cationicgroups, and whose hydrophobic part consists of any other polymer, suchas polyisobutylene or polypropylene oxides.

When the emulsions of this invention are to be used in artificial bloodsor red blood cell substitutes, the surfactant, or combinations of them,must be physiologically acceptable. For example, in artificial bloods analkylphosphoryl choline or dialkylglycerophosphoryl choline is usedwhere the alkyl group is about C₁₂ -C₁₈ as exemplified by1-octadecyl-2-hexadecylglycero-3-phosphoryl choline.

The emulsions may be prepared using any order of mixing the maincomponents of oil, surfactant and water. However, for optimal PFCemulsions the PFC is first mixed with the adjuvant oil in the presenceof a combination of all or part of the surfactant and some water. Thenthe final emulsion is prepared by emulsifying this first emulsion in theremaining water and any remaining surfactant as described in the aboveClark and Shaw European Appln. 87300454.3 which is incorporated hereinby reference.

The mixing and emulsification of components may be done using any of theconventional mixers and emulsifiers. For example, Fisher brand touchmixers, Microfluidizers, Gaulin and Rannie Homogenizers may be employed.

The following non-limiting examples illustrate various embodiments ofthis invention.

DETAILED EXAMPLES Surfactant Synthesis

The surfactants of this invention were synthesized from thecorresponding dialkylglycerols and the synthetic sequences arerepresented by the following schemes or methods. ##STR8##

In connection with the above schemes and methods, the term "symmetrical"is employed to designate dialkyl compounds where both alkyl groups areidentical and "unsymmetrical" where the alkyl groups differ. Employingthe schemes and methods, the following illustrate specific examples ofmaking dialkyletherglycerols as precursers of thedialkylglycerophosphoryl choline surfactants of this invention.

3-Benzyl-1,2-dihexadecylglycerol (Scheme Ia)

To a refluxing mixture of benzylglycerol (50 g, 0.27 mol) and KOH (40 g,0.76 mol), which had been freshly powdered in an electric millingdevice, in toluene (500 mL) was added 1-bromohexadecane (252 g, 9.76mol) over a period of one hour. The mixture was refluxed overnight, withDean-Stark removal of water of reaction. The slurry was stripped oftoluene on a rotary evaporator, then the residue was slurried in CHCl3and washed with water. The organic phase was dried (MgSO4) and stripped.Unreacted starting material was removed by Kugelrohr distillation (150°,1 mm Hg). The pot residue was a yellow solid of sufficient purity foruse in the next step.

1,2-Dihexadecylglycerol (Scheme Ib)

The residue from the Kugelrohr distillation was transferred to a Parrhydrogenator and dissolved in EtOAc (200 ml) and HOAc (200 ml). Undernitrogen, 5% Pd/C (Sg) was added. Debenzylation was carried out at 50°C. and 50 psig until hydrogen uptake had ceased. The reaction mass wasfiltered warm and the filter cake washed with chloroform. Afterevaporation of the volatile components on a rotary evaporator theresidue was recrystallyzed from ethyl acetate to yield white crystals(200 g, 69%) m.p. 56°-7° C.

Other compounds prepared by Scheme I:

1,2-dioctadecylglycerol*

1,2-ditetradecylglycerol

1,2-dioctylglycerol

1,2-didecylglycerol

*This compound may also be named 2,3-bis(octadecyloxy)-1-propanol andthis nomenclature may apply to the other compounds.

2-Bromoethylphosphoryl dichloride

To a mixture of freshly distilled phosphorus oxychloride (62 ml) in 50ml carbon tetrachloride was added 62 g of bromoethanol, dropwise withstirring at 20° C. The mixture was allowed to stand over night and thenheated at reflux for 2 hours to drive off hydrogen chloride. Afterstripping the carbon tetrachloride the residue was fractionated invacuo. Yield 70-77g, b.p. 110°-115° C. @12 mm Hg.

2-Bromoethylester of 1,2-dihexadecylglycerol phosphoric acid (Method Ac)

2-bromoethylphosphoryl dichloride (14.5 g, 0.074 ml) was dissolved inanhydrous ether (300 ml) and triethylamine (7.5 g, 0.074 mol). To thissolution was added (20 g, 0.037 mol) of dihexadecylglycerol at once atroom temperature. The reaction mixture was allowed to stir over night.Hydrolysis of the intermediate was carried out by vigorous stirring withH₂ O (300 ml) and MeOH (100 ml) for 5 hours. The layers were allowed toseparate and the ether layer was dried over MgSO4. After removing theether on a rotary evaporator 26.3 g of white waxy solid was obtained.

1,2-Dihexadecylglycerophosphoryl choline (Method A)

To a solution of the above 2-bromoethylester of Method A (82 g, 0.18mol) in methyl ethyl ketone (400 ml) was added trimethylamine (35 g, 0.6mol) in a sealed vessel, which was then heated to 55° C. for 18 hours.The slurry was then cooled to room temperature, filtered and the cakewashed with acetone. The cake was dissolved in hot 90% ethanol (1400ml), Dowex SBR (200 g) ion exchange resin was added and the slurrystirred for 15 minutes. The resin was filtered off and ethanol-waterremoved on a rotary evaporator. The residue was dried over P2O5 and thenpurified by column chromatography to give 35.5 g (42.2% yield) of pureproduct.

1,2-Dioleylglycerophosphoryl choline (Method B)

To a thoroughly dried 500 ml round bottom flask equipped with condenser,stirrer, and thermometer, was added dioleyl glycerol (Scheme 1bhereinafter) (40 g, 67 mmol) and anhydrous diethyl ether (380 ml). Thesolids were dissolved by mild warming, then the solution was cooled toroom temperature. A solution of 2-chloro-2-oxo-1,3,2-dioxaphospholane(9.8 g, 69 mmol) in diethyl ether (50 ml) was added dropwise, over thespace of 15 minutes. At that point, triethylamine (TEA) (7.2 g, 71 mmol)was added, and a white precipitate formed almost immediately. Themixture was stirred for one hour, then filtered under a stream of drynitrogen. The filtrate was rotary evaporated to obtain a white solid.This solid was dissolved in acetonitrile (400 ml) in a 500 ml serumbottle, and trimethylamine (TMA) (28.0 g, 237 mmol) was added. Thebottle was sealed and kept at 65° C. for 18 hours. After cooling to roomtemperature, the contents were dissolved in CHCl 3 and the solvents wereevaporated to yield a white solid, which was chromatographed on silicagel (75% CHCl13, 22% H₂ O, 3% H₂ O eluent) to yield the desired product(21.2 g, 41.5% from the starting dioleylglycerol). (Note: Method B wasthe method of choice for incorporating the phosphoryl headgroup.)

1,2-Dioleylglycerol (Scheme Ib)

A 250 ml flask equipped with a dry ice condenser and a dry ice-acetonebath was charged with anhydrous NH3 (140 ml). After cooling to -65° C.under N2 Li wire (1.35 g) was added in small pieces.Dioleylbenzylglycerol (10 g, 0.0146 mol) in THF (60 ml) and EtOH (12 g)was added over 30 minutes and then stirred at -65° C. for 15 minutes.The dry ice bath was removed and the reaction mass allowed to warm toroom temperature, with evaporation of NH3. Water (100 ml) and heptane(150 ml) were added. The organic phase was washed with water, dried(MgSO4) and stripped of solvent yielding a light yellow oil (7.8 g,90%).

1-hexadecyl-2-tetradecyl glycerol (Scheme II c, d, e)

1-hexadecyl glycerol (105g, 0.33mol) and 93 g (0.33mol) tritylchloridewere dissolved in 300 ml of pyridine. The mixture was heated at 100° C.for 16 hours. After cooling to room temperature 1500 ml of ethyl etherwas added and the slurry washed 3 times with cold 0.5N H2SO4, 5% sodiumbicarbonate and 2 times with water. The organic phase was dried overMgSO4, the ether removed on a rotary evaporator and the residuerecrystallized from ethanol, yielding the intermediate1-hexadecyl-3-trityl glycerol (129 g, 70%). The 2 position of thisintermediate was alkylated with tetradecyl bromide in the same manner(powdered KOH in toluene) as described earlier for the dialkylation ofbenzyl glycerol in the preparation of 1,2-dihexadecyl glycerol. Theresulting 1-hexadecyl-2-tetradecyl-3-trityl glycerol (128 g) wasdissolved in 300 ml of petroleum ether. At room temperature 30 g ofanhydrous hydrogen chloride was added. A precipitate formed within ashort time. The slurry was stirred at room temperature for 4 hours andthen filtered. The solids were recrystallized from acetone (10° C.) togive 45.6g of product, m.p. 42°-3° C.

Hemolysis Screening of Surfactants

Surfactant Examples 1a-1k according to general Structure I above, and 2aof Structure II above, were prepared following the above procedures.Hemolysis screening of these surfactants is reported in the followingTable I.

Surfactants were screened initially by preparing 5%solutions/dispersions of the surfactant in 0.9% saline and admixing 1:1with rat whole blood. Centrifugation (6 minutes) at 2400 rpm affordedpacked cells and a supernate. The surfactant was judged to be hemolyticat the given concentration if the supernate from the centrifugationshowed a visually distinct red hue relative to control rat blood. Thediscoloration of the supernate was not quantified colorimetrically. Inthe preliminary studies, low molecular weight surfactants 1a and 1b werefound to be hemolytic upon mixing with whole red blood and were notsubjected to further toxicity testing, thus "N/A" was used in Table I.Those compounds that were found to be non-hemolytic were then tested ina preliminary acute toxicity screen by injection of 40 cc/Kg of a 5%sonicated dispersion of the compound in 0.9% saline into two SpragueDawley rats. The emulsion passed toxicity testing if the animalssurvived for 14 days, and these results are reported in Table I. Whilethe study tended to demonstrate that the low molecular weight compoundswere hemolytic and therefore toxic, whereas larger C₁₄ -C₁₈ groups werenonhemolytic, further testing may be required to rationalize differencesin lethality.

                  TABLE I                                                         ______________________________________                                        HEMOLYSIS SCREENING OR SURFACTANTS                                                                                 Survival at                                                                   40 cc/Kg of                              Cpd  R.sub.1  R.sub.2 R.sub.3                                                                              Hemolytic                                                                             5% solution                              ______________________________________                                        1a   C.sub.8 H.sub.17                                                                       C.sub.8 H.sub.17                                                                             Yes     N/A                                      1b   C.sub.14 H.sub.29                                                                      C.sub.14 H.sub.29                                                                            Yes     N/A                                      1c   C.sub.16 H.sub.33                                                                      C.sub.16 H.sub.33                                                                            No      2/2                                      1d   C.sub.18 H.sub.37                                                                      C.sub.18 H.sub.37                                                                            No      insoluble cpd                            1e   C.sub.16 H.sub.33                                                                      C.sub.14 H.sub.29                                                                            No      0/2                                      1f   C.sub.18 H.sub.37                                                                      C.sub.14 H.sub.29                                                                            No      2/2                                      1g   C.sub.14 H.sub.29                                                                      C.sub.18 H.sub.37                                                                            No      0/2                                      1h   C.sub.16 H.sub.33                                                                      C.sub.18 H.sub.37                                                                            No      0/2                                      1i   C.sub.18 H.sub.35                                                                      C.sub.18 H.sub.35                                                                            No      2/2                                      1j   C.sub.18 H.sub.37                                                                      C.sub.16 H.sub.33                                                                            N/A     N/A                                      1k   C.sub.14 H.sub.29                                                                      C.sub.16 H.sub.33                                                                            N/A     N/A                                      2a                    C.sub.32 H.sub.65                                                                    No      2/2                                      ______________________________________                                    

PERFLUOROCARBON EMULSIONS AND THEIR PROPERTIES

The surfactants of Table I were used to make 40 volume % (v/v)perfluorochemical emulsions. In the above described European Pat. Appln.87300454.3, it was reported that certain oils, i.e., triglycerides offatty acids as co-additives greatly improved the stability of lecithincontaining emulsions. Therefore, those perfluorochemical emulsions wereused as a control for comparison with the perfluorochemical emulsions ofthis invention. The control emulsion was a 40 v/v % perfluorooctylbromide emulsion prepared according to the technique of the mentionedEuropean patent application and containing 1.75 w/v % egg yolk lecithinas the surfactant and 2 w/v % safflower oil. The surfactants of theinvention were substituted for lecithin and the results of theemulsion's properties are reported in Table II. In Table II, theemulsion compositions, mean particle size as measured by laser lightscattering, and stability toward storage in a 5% solution of SerumAlbumin in Lactated Ringer's solution after 4 days at 37° C. arereported. All data is for emulsions sterilized for 15 minutes at 121° C.in a rotating basket autoclave, the industry standard for large volumeparenterals.

                  TABLE II                                                        ______________________________________                                        EMULSIONS AND PROPERTIES                                                              w/v %   w/v %    Mean Particle                                                                           4 days                                     Cpd.    Cpd.    oil      Size (nM) Serum (nM)                                 ______________________________________                                        1a      2.0     0.0      235       249                                        1c      4.0     0.0      212       --                                                 1.5     2.0      270       267                                        1e      1.5     2.0      347       385                                        1f      2.0     2.0      359       357                                        1g      2.0     2.0      392       438                                        1h      2.0     2.0      235        9                                         1i      1.8     2.0      304       258                                        2a      1.8     2.0      275       254                                        Control 1.75    2.0      250       260                                        ______________________________________                                    

For comparison with the data of Table II, the study was repeated with aconstant loading of 2 w/v % surfactant, and 2 w/v % safflower oiladjuvant for the control and surfactants of this invention.

                  TABLE III                                                       ______________________________________                                        EMULSIONS AND PROPERTIES                                                                   Mean Particle                                                                             4 days                                               Cpd          Size (nM)   Serum (nM)                                           ______________________________________                                        1c           421         318                                                  1e           256         256                                                  1f           333         320                                                  1g           207         N/A                                                  1h           354         460                                                  1i           323         301                                                  1j           229         N/A                                                  1k           227         N/A                                                  Control      226         232                                                  ______________________________________                                    

With reference to the data of Tables I and II, the emulsions displayedvery good particle size distribution characteristics as measured by alaser light scatterer, both at the time of preparation ("Mean ParticleSize" column) and after 4 days storage at 37° C. in a mixture of HumanSerum Albumin and Lactated Ringer's Solution ("Serum" column) designedto mimic blood and plasma. All data point to surprisingly stableemulsions when the surfactants of this invention were substituted foregg yolk lecithin.

IMPACT OF FORMULATION ON PERFLUOROCHEMICAL CIRCULATORY BLOOD RESIDENCETIME

The emulsions of Tables II and III were studied in a randomized, tenanimal per emulsion experiment in which each animal was infused with 20cc/Kg of emulsion and sacrificed at 48 hours to determine thecirculatory perfluorochemical or perfluorocarbon content called"fluorocrit" and abbreviated "Fct". The Fct represents that percentageof the blood that is perfluorocarbon in v/v %.

Two sets of ten rats each were treated with a control emulsion and anemulsion prepared from the surfactant of this invention using arandomized administration design. After 48 hrs., the animals wereanesthetized, the chest cavity was opened, and a blood sample (1-2ml)was withdrawn from the still beating heart. Three to 4 drops of thesample were added to each of three previously tared 4 ml vialscontaining 10%KOH/EtOH (1.0 ml). The vials were weighed to determine theamount of blood added, then reweighed after an internal standard mixtureof 1% octane in CFCl3 (1.5-2.0ml) was added. H₂ O was added to the vialuntil full, then the vial was placed on a rotary shaker at 240 rpm for20 minutes. The lower layer was withdrawn, and passed through a glasswool plugged pasteur pipette containing silica gel (0.3-0.4g). Theeluate was collected in Hewlett Packard autosampler vials and injectedonto a 5890A chromatograph containing a 30 meter, 0.054" ID Megabore,0.3 coated DB-1 column. The program parameters specified a hold periodat 40° C. for 4 minutes, followed by a 10° C./minute temperature riseuntil 140° was attained.

The impact of the formulation on fluorochemical circulatory bloodresidence time is reported in Table IV.

                  TABLE IV                                                        ______________________________________                                        PERFLUOROCHEMICAL CIRCULATORY BLOOD                                           RESIDENCE TIME                                                                              48 hr                                                                  Cpd    Fct @ 20 cc                                                     ______________________________________                                               1c     6.18                                                                   1e     6.72                                                                   1f      8.18*                                                                 1g     7.22                                                                   1h     N/A                                                                    1i     4.93                                                                   1j      8.65*                                                                 1k     5.38                                                                   Control                                                                              6.46                                                            ______________________________________                                         *significantly differs from control (Fisher Protected Least Significant       Difference) at the 99% confidence Level                                  

With reference to Table IV, it is apparent that emulsions made with thesurfactants of this invention, especially as represented by compounds 1fand 1j, show increased stabilities in the circulatory blood system asmeasured by the amount of perfluorochemical remaining at 48 hours.Therefore, in addition to the stable PFC emulsions formed by thesurfactants of this invention, they significantly increase thecirculatory blood residence time of the PFC.

IMPACT OF FORMULATION ON ORGAN DISTRIBUTION OF PERFLUOROCHEMICAL

The impact of the emulsions of Table IV on organ distribution of PFC wasthen determined by sacrificing the test animals 14 days after infusion.The results of the organ analysis testing are shown in Table V.

                  TABLE V                                                         ______________________________________                                        ORGAN DISTRIBUTION 14 DAYS AFTER                                              INFUSION OF 40 cc/Kg                                                                Liver    Spleen   Lung   Kidney       Sur-                              Cpd   g/100 g  g/100 g  g/100 g                                                                              g/100 g                                                                              Hct   vival                             ______________________________________                                        1e    7.97     1.55**   0.57*  1.06   35.8  9/10                              1g    7.52*    1.49**   0.55*  0.94   39.0  6/10                              1i    7.47*    2.56     0.64*  0.96   34.7  8/10                              1j     6.64**  1.66**    0.57**                                                                              0.89   44.4**                                                                              9/10                              1k    7.76*    2.09*    0.64*  1.22   36.5  9/10                              Con-  9.13     2.89     0.87   1.02   31.7  9/10                              trol                                                                          ______________________________________                                         *Significantly different from control at 99% confidence level by Fisher       Protected Least Significant Difference                                        **Significantly different from control at 99% confidence level by Fisher      Protected Least Significant Difference and by Scheffe Ftest              

It is clear from Table V that the surfactant compounds of this inventiongreatly ameliorate the effects of perfluorochemical on the toxicresponses of hepatomegaly, splenomegaly and lung enlargement.Furthermore, the surfactants do not cause nearly as large a drop inhematocrit (the v/v % of red blood cells--"Hct") after infusion. Theresult was so striking in the case of compound 1j that it was pursued intwo additional separate studies as reported in Tables VI and VII.

                  TABLE VI                                                        ______________________________________                                        1-OCTADECYL-2-HEXADECYLGLYCERO-3-                                             PHOSPHORYL CHOLINE SURFACTANT                                                       Liver    Spleen   Lung   Kidney       Sur-                              Cpd   g/100 g  g/100 g  g/100 g                                                                              g/100 g                                                                              Hct   vival                             ______________________________________                                        1j    7.44     1.70*    0.98   0.97   36.1  10/10                             Con-  7.99     2.51     0.92   0.93   32.1**                                                                               8/10                             trol                                                                          ______________________________________                                         *Significantly different from control at 97.4% probability by Student's t     **Significantly different from control at 99.3% probability by Student's      t.                                                                       

                  TABLE VII                                                       ______________________________________                                        1-OCTADECYL-2-HEXADECYLGLYCERO-3-                                             PHOSPHORYL CHOLINE SURFACTANT                                                       Liver    Spleen   Lung   Kidney      Sur-                               Cpd   g/100 g  g/100 g  g/100 g                                                                              g/100 g                                                                              Hct  vival                              ______________________________________                                        1j    7.39     1.46*    0.828  0.901  37.7 10/10                              Con-  7.82     2.78     0.838  0.943  34.1 10/10                              trol                                                                          ______________________________________                                         *Significantly different from control at 99% probability by Student's t. 

Tables VI and VII demonstrate that splenomegaly was reduced andcirculating red cell count as measured by hematocrit remained high. Theeffects on the liver and lung were not as pronounced in Table VI as inTable V. However, with respect to Table VII, it is believed that thesmaller values obtained for the liver and spleen are also real. Eventhough the t-test does not show them to be significantly different inthe individual studies at the 99% level, the liver weights remainsmaller than the control, and the student t probability of a differencehas been 86%. Accordingly, the odds of these results occurring in threeseparate experiments are very low. In addition, blood hematology wasmonitored by accredited outside laboratories and the results are shownin Table VIII.

                  TABLE VIII                                                      ______________________________________                                        BLOOD HEMATOLOGY                                                                               Hemo-              Reticulo-                                 Cpd       RBC    globin      Platelets                                                                            cytes                                     ______________________________________                                        1j        5.42*  11.04*      1024222                                                                              313281**                                  Control   4.80   9.79         920300                                                                              481858                                    ______________________________________                                         *Significantly different from control at 99% probability by Student's t.      **Significantly different from control at 98% probability by Student's t.

With reference to Table VIII, these results demonstrate conclusivelythat increased circulating red cell count is real. With reference to theabove detailed description and experimental results, the benefits ofthis invention may be appreciated. In summary, a new class ofsurfactants has been found to stabilize oil and water emulsions in abroad sense. The stability has been particularly demonstrated in thecase of emulsions containing oils of triglycerides of fatty acids asemulsified particles and oleophilic perfluorochemicals as emulsifiedparticles. In addition to the surprisingly stable PFC emulsions, thesurfactants significantly increased the circulatory residence time ofthe PFC in animal blood, favorably altered the tissue distribution ofthe PFC in critical organs, and significantly ameliorated an adversedrop in red cell count.

The surfactants of this invention are also resistant to oxidation anddegradation normally associated with egg yolk phospholipid lecithinemulsifying agents. Accordingly, emulsions containing the novelsurfactants may be oxygenated during sterilization and through storagefor extended periods without degradation due to these oxygen resistantsurfactants.

In view of the above detailed description, other variations andembodiments of this invention will be understood to a person of ordinaryskill in this art and such are within the scope and spirit of thisdescription.

What is claimed is:
 1. A red blood cell substitute comprising an amountof a physiologically acceptable emulsion of a fluorochemical, water anda surfactant having a general structure of ##STR9## where R₁ and R₂ area C₄ -C₈ saturated or unsaturated aliphatic group and PC is thephosphoryl choline group or salt thereof represented by the structure##STR10## where R₄ is hydrogen or lower alkyl from the group consistingof methyl, ethyl and propyl, said amount being therapeutically effectivefor oxygen carrying and transport in animals.
 2. The red blood cellsubstitute of claim 1 where R₄ is methyl.
 3. The red blood cellsubstitute of claim 1 wherein said surfactant is selected from the groupconsisting of 1,2 -ditetradecylglycero-3-phosphoryl choline,1,2-dihexadecylglycero-3-phosphoryl choline,1,2-dioctadecylglycero-3-phosphoryl choline,1-hexadecyl-2-tetradecylglycero-3-phosphoryl choline,1-octadecyl-2-tetradecylglycero-3-phosphoryl choline,1-tetradecyl-2-octadecylglycero-3 -phosphoryl choline,1-hexadecyl-2-octadecylglycero-3 -phosphoryl choline, 1,2-octadecylglycero-3-phosphoryl choline,1-octadecyl-2-hexadecylglycero-3-phosphoryl choline,1-tetradecyl-2-hexadecylglycero-3-phosphoryl choline, and1-hexadecyl-2-tetradecylglycero-3-phosphoryl choline.
 4. The red bloodcell substitute of claim 1 wherein the fluorochemical is selected fromthe group consisting of perfluorodecalin, perfluoromethyldecalin,perfluorodimethyldecalin, perfluorodimethyladamantane,perfluorooctylbromide, perfluoro-4-methyloctahydroquinolidizine,perfluoro-N-methyldecahydroquinoline, F-methyl-1-oxadecalin,perfluorobicyclo(5.3.0)-decane, perfluorooctahydroquinolidizine,perfluoro-5,6-dihydro-5-decene, and perfluoro-4,5-dihydro-4-octene,chlorinated perfluorocarbons, and mixtures thereof.
 5. The red bloodcell substitute of claim 4 that is stable after heat sterilization. 6.The red blood cell substitute of claim 4 wherein a liquid fatty oil ispresent as an emulsifying adjuvant in an amount between 0.5 and about30% by weight of the emulsion.
 7. The red blood cell substitute of claim6 wherein the oil is selected from the group consisting of mono-, di-and triglycerides, and mixtures thereof.
 8. The red blood cellsubstitute of claim 1 wherein the surfactant is present in an amountfrom about 0.5 to about 10% by weight of the emulsion.
 9. The red bloodcell substitute of claim 1 wherein the surfactant is present in anamount of from about 1 to about 2% by weight of the emulsion.
 10. Thered blood cell substitute of claim 1 wherein the fluorochemical ispresent in an amount of from about 10 to about 75% by volume of theemulsion.
 11. The red blood cell substitute of claim 4 wherein saidsurfactant is selected from the group consisting of 1,2-ditetradecylglycero- 3 -phosphoryl choline,1,2-dihexadecylglycero-3-phosphoryl choline,1,2-dioctadecylglycero-3-phosphoryl choline,1-hexadecyl-2-tetradecylglycero-3-phosphoryl choline,1-octadecyl-2-tetradecylglycero-3-phosphoryl choline,1-tetradecyl-2-octadecylglycero-3-phosphoryl choline,1-hexadecyl-2-octadecylglycero-3-phosphoryl choline,1,2-dioctadecylglycero-3-phosphoryl choline,1-octadecyl-2-hexadecylglycero-3-phosphoryl choline,1-tetradecyl-2-hexadecylglycero-3-phosphoryl choline, and1-hexadecyl-2-tetradecylglycero-3-phosphoryl choline.
 12. The red bloodcell substitute of claim 1 wherein a fluorochemical is contained in anamount of from about 10 to about 60% by volume of the emulsion.
 13. Thered blood cell substitute of claim 1 wherein said flurochemical iscontained in an amount of at least about 40% by volume of the emulsion.14. The red blood cell substitute of claim 1 wherein said surfactant is1-octadecyl-2-hexadecylglycero-3-phosphoryl choline.
 15. The method ofincreasing the fluorochemical content of circulating blood in an animalfor oxygen carrying and transport by administering the emulsion ofclaim
 1. 16. The method of increasing the fluorochemical content ofcirculating blood in an animal for oxygen carrying and transport byadministering the emulsion of claim
 3. 17. The method of increasing thefluorochemical content of circulating blood in an animal for oxygencarrying and transport by administering the emulsion of claim
 4. 18. Themethod of claim 15 conducted by intravenous infusion of said emulsion.19. The method of claim 15 conducted without a drop in the hematocrit ofsaid animal blood.
 20. The method of claim 15 conducted to reduceresidual distribution of said fluorochemical in the organs of saidanimal.